Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans

UDP-Glucose: Glycoprotein Glucosyltransferase 1,2 (UGGT1,2)

Almost one-third of proteins synthesized by eukaryotic cells belong to the secretory pathway, entering the endoplasmic reticulum (ER) either co- or posttranslationally. In the ER, proteins acquire their native tertiary fold, disulfide bonds are formed, and in some cases, they assemble into oligomeric structures. Numerous folding-assisting enzymes and chaperones are in place to ensure the efficiency of these processes. Additionally, almost 70 % of the secretory pathway proteins are N-glycosylated by the translocon-associated oligosaccharyltransferase complex in the consensus sequence Asn-X-Ser/Thr, in which X can be any amino acid except for Pro (Apweiler et al. 1999). The consensus sequences are N-glycosylated as they emerge into the ER lumen when there are about 12–13 amino acids between the Asn residue and the inner ER membrane surface. In some cases, the same modification may occur posttranslationally (Ruiz-Canada et al. 2009). N-glycosylation is one of the most abundant and relevant protein modifications as N-glycans are central players in molecular recognition events, a function particularly suitable for them given their diverse composition. Additionally, N-glycans may modulate the biophysical behavior of their protein moieties. N-glycans may inhibit protein aggregation, may increase resistance to proteolytic degradation, and may promote acquisition of elements of secondary structure such as turns (Chen et al. 2010). Of particular relevance is the involvement of N-glycans in glycoprotein folding in the ER (Caramelo and Parodi 2007; D’Alessio et al. 2010). In this case, N-glycans act as an epigenetic information platform indicating the folding status of glycoproteins. This information is generated by glycosyltransferases and glycosidases that translate the conformational status of glycoproteins into particular N-glycan structures. Upon their recognition, ER-resident lectins retain the immature species in the ER, thus promoting their proper folding and hindering their Golgi exit. N-glycosylation starts in most eukaryotic cells with the transfer of the entire glycan Glc3Man9GlcNAc2 from a dolichol-P-P derivative.Fil: Parodi, Armando José A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: D'alessio, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Structural Insights into the Broad-Spectrum Antiviral Target Endoplasmic Reticulum Alpha-Glucosidase II

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme’s α- and β-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins